Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities

Abstract

We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor.

Keywords: Adenylyl cyclase (AC activity); Cannabinoid receptor subtype 2 (CB2); Cyclic adenosine monophosphate (cAMP); Molecular modeling; Site-directed mutagenesis; Time-resolved fluorescence resonance energy (TR-FRET) transfer.

Fig.1

Molecular docking of CB2 ligands into putative CB2 binding site. A. 3D homology model of the CB2 seven transmembrane helices showing amphipathic-binding cavity, lipophilicity scale is used to colored the pocket in which hydrophobic areas are shown in brown and hydrophilic areas are in blue. B–D, Interactions between each ligand and the receptor. Magnified views of the predicted binding pocket, which show hydrophobic residues such as L201, V261, V113, L192 showed interact with CP-55,940, SR144528 and XIE95-26. In addition, N188 reveal H-bond with both CP-55,940 and SR144528.

Fig.2

Western Blot analysis of wild-type and mutant h-CB2 receptor isoforms in transfected CHO cells. From left to right: 1- CB2 WT (wild-type), 2- V113E, 3- V113L 4- L192S, and 5- L192A.

Fig.3

Competition binding assay for wild-type CB2 (■) and mutants receptors, CB2 V113L (▼), L192A (●) binding to (A. CP-55,940; B. SR144528; C. XIE95-26). The assays were performed to the membranes prepared from CHO cell line stably expressing wild-type or mutants human CB2receptors. Data are mean ± S.E.M. of two independent experiments performed in duplicate. Curves were generated using GraphPad Prism 5.

Fig.4

Agonist-induced inhibition of forskolin-stimulated cAMP accumulation of CB2 WT and all mutants’ receptors. (▼) WT- CB2, (▲) V113L, (■) V113E (●) L192A and (◆) L192S. All receptors were stably expressed in CHO cells. Data are mean ± S.E.M. of at least three independent experiments in triplicate.